Synthesis and α-glucosidase inhibitory mechanisms of bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, a potential marine bromophenol α-glucosidase inhibitor

Article abstract of DOI:10.3390/md9091554

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE), derived from the marine algae, is a potential α-glucosidase inhibitor for type 2 diabetes treatment. In the present study, a synthetic route was established as a valid approach to obtain BDDE. Fluorescence spectra, circular dichroism spectra and molecular docking methods were employed to elucidate the inhibitory mechanisms of BDDE against α-glucosidase. The results showed that BDDE could be prepared effectively and efficiently with the established synthetic methods. Synthetic BDDE bound with α-glucosidase and induced minor conformational changes of the enzyme. The docking results indicated the interaction between BDDE and α-glucosidase was driven by both hydrophobic forces and hydrogen bonds. The docked BDDE molecule was completely buried in the α-glucosidase binding pocket with part of the molecule reaching the catalytic center and overlapping with the position of glucose, and the rest of the molecule extending towards protein surface. This study provides useful information for the understanding of the BDDE-α-glucosidase interaction and for the development of novel α-glucosidase inhibitors.

Full text of DOI:10.3390/md9091554

Mar. Drugs 2011, 9, 1554-1565; doi:10.3390/md9091554
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Synthesis and α-Glucosidase Inhibitory Mechanisms
of Bis(2,3-dibromo-4,5-dihydroxybenzyl) Ether, a Potential
Marine Bromophenol α-Glucosidase Inhibitor
Ming Liu 1, Wei Zhang 2, Jianteng Wei 1 and Xiukun Lin 1,*
1
Institute of Oceanology, Chinese Academy of Science, 7 Nanhai Rd, Qingdao 266071, China;
E-Mails: lmouc@hotmail.com (M.L.); weijt@163.com (J.W.)
2
Southern Research Institute, 2000 9th Avenue South, Birmingham, AL 35205, USA;
E-Mail: zhangwee@yahoo.com
* Author to whom correspondence should be addressed; E-Mail: linxiukun@yahoo.com;
Tel./Fax: +86-532-8289-8916.
Received: 1 July 2011; in revised form: 26 July 2011 / Accepted: 8 August 2011 /
Published: 19 September 2011
Abstract: Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE), derived from the marine
algae, is a potential α-glucosidase inhibitor for type 2 diabetes treatment. In the present
study, a synthetic route was established as a valid approach to obtain BDDE. Fluorescence
spectra, circular dichroism spectra and molecular docking methods were employed to
elucidate the inhibitory mechanisms of BDDE against α-glucosidase. The results showed
that BDDE could be prepared effectively and efficiently with the established synthetic
methods. Synthetic BDDE bound with α-glucosidase and induced minor conformational
changes of the enzyme. The docking results indicated the interaction between BDDE and
α-glucosidase was driven by both hydrophobic forces and hydrogen bonds. The docked
BDDE molecule was completely buried in the α-glucosidase binding pocket with part of
the molecule reaching the catalytic center and overlapping with the position of glucose, and
the rest of the molecule extending towards protein surface. This study provides useful
information for the understanding of the BDDE-α-glucosidase interaction and for the
development of novel α-glucosidase inhibitors.
Keywords: bromophenol; bis(2,3-dibromo-4,5-dihydroxybenzyl) ether; α-glucosidase inhibitor

Mar. Drugs 2011, 9
1555
1. Introduction
α-Glucosidase, is involved in carbohydrate synthesis and breakdown, plays crucial role in diabetes,
viral infection and cancer. Due to its diverse bioactivities, α-glucosidase has been considered to be a
preferred drug target in the pharmaceutical area and a number of α-glucosidase inhibitors have been
discovered and studied. The clinically used anti-diabetic agents acarbose [1], voglibose, and
miglitol [2], inhibit α-glucosidase competitively in the brush border of the small intestine, and
consequently delay the hydrolysis of carbohydrates and alleviate postprandial hyperglycemia.
However, the continuous administration of these agents may cause some adverse effects, such as
diarrhoea, abdominal discomfort, flatulence [3–5], and hepatotoxicity [6]. It is therefore still necessary
to develop novel α-glucosidase inhibitors. The therapeutic challenge of type 2 diabetes mellitus
promotes the search for novel α-glucosidase inhibitors and a number of them have been
investigated [7–11].
One such newly reported group of α-glucosidase inhibitors are the marine natural
bromophenols [12–15], which are usually isolated from the marine algae. As the α-glucosidase
inhibitors, these bromophenols have been reported to inhibit against α-glucosidase with low IC50’s
(Table 1), and this activity has a close relationship with the Br and phenolic unit in the molecules. One
of the most potent bromophenol α-glucosidase inhibitors is bis(2,3-dibromo-4,5-dihydroxybenzyl)
ether (BDDE, Figure 1), which competitively inhibits α-glucosidase with an IC50 of 0.098 μM [12,13,15].
BDDE has also been reported to possess anticancer [16], and antibacterial activities [17]. The previous
promising research results indicate that BDDE represents a novel lead compound for the design of new
drugs. However, low content in marine algae as well as the difficulty in extraction and separation have
hindered further investigations of BDDE bioactivities. In addition, few efforts have been contributed to
explore the interaction between BDDE and α-glucosidase, which is important for dissecting the
structure-activity relationship for further optimizations.
In this study, we present a synthetic route that can provide sufficient amounts of BDDE to facilitate
bioactivity studies, and we further investigate the mechanisms underlying BDDE inhibition using
fluorescence spectra, circular dichroism (CD) spectra, and molecule docking.
Table 1. IC50 values and S. cerevisiae α-glucosidase inhibition modes of bromophenols
purified from different marine algae.
Marine bromophenol
(μM) Inhibition mode Reference
Bis(2,3,6-tribromo-4,5-dihydroxybenzyl) ether
Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether
2,3,6-Tribromo-4,5-dihydroxybenzyl alcohol
0.03
Unknown
[13]
[12,13,15]
[15]
0.098 Competitive
11
Mixed
4-Bromo-2,3-dihydroxy-6-hydroxymethylphenyl-2,5- 25
dibromo-6-hydroxy-3-hydroxymethylphenyl ether
Unknown
[12]
2,4,6-Tribromophenol
60.3 Mixed
[14]
[12]
[13]
[14]
2,3-Dibromo-4,5-dihydroxybenzyl alcohol
3-Bromo-4,5-dihydroxybenzyl alcohol
2,4-Dibromophenol
89
100
Mixed
Mixed
110.4 Mixed

Mar. Drugs 2011, 9
1556
Figure 1. Structure of bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE).
Br
Br
Br
Br
O
HO
OH
OH
OH
2. Results and Discussion
2.1. Synthesis of BDDE
The abundance of BDDE in the fresh red algae is very low (about 3 × 10−3% W/W) [12], which
makes further investigation difficult. In order to obtain sufficient quantities of pure BDDE, we initiated
development of an improved synthesis efforts on BDDE. The synthetic route established, shown in
Scheme 1, was different from the published synthetic methods [18]. Although this route is four steps
longer, with a total yield of 4.7%, the workup process is convenient and this route avoids
the instability of the intermediate products and also avoids the production of byproduct
(2-bromo-4,5-dihydroxybenzyl)(2,3-dibromo-4,5-dihydroxybenzyl) ether, which might complicate the
purification process. Therefore, it is an alternate efficient approach to obtain the target compound. The
1
structure of target compound 7 was confirmed by the following H NMR spectroscopic analysis and
1
compared with the data in published literature [12,15]: H NMR (400 MHz, DMSO) δ: 4.344 (4H, s,
H-7, 7′), 6.939 (2H, s, H-6, 6′), 9.567 (2H, s, HO-5, 5′), 10.085 (2H, s, HO-4, 4′). Compound 7
exhibited inhibitory activity against α-glucosidase with an IC50 value of 0.116 μM, which was
consistent with BDDE isolated from marine algae [12,13,15]. Therefore, we established a valid
synthesis route and provided enough amount of BDDE for the following studies both in vitro and
in vivo, and also for the evaluations of its other bioactivities.
Scheme 1. Reagents and Conditions: a: AcOH, Br2, r.t. 2 h; b: MeI, K2CO3, CH3CN, r.t.,
4 h; c: AcOH, Br2, 60 °C overnight; d: NaBH4, MeOH, −20 °C to r.t., 3 h; e: TsCl, Et3N,
CH3CN, reflux overnight; f: BBr3, CH2Cl2, 0 °C to r.t., 3 h.
Br
Br
Br
O
Br
O
O
c
d
a
O
b
HO
HO
O
O
O
O
O
O
4
1
2
3
Br
Br
Br
Br
Br
Br
Br
Br
O
Br
Br
e
OH
O
f
O
O
O
HO
OH
O
O
O
OH
OH
5
6
7

Mar. Drugs 2011, 9
1557
2.2. BDDE Binding Quenched the Intrinsic Fluorescence of α-Glucosidase
The previously reported competitive inhibition on α-glucosidase in vitro [15] suggests that BDDE
may directly bind to the active site of the enzyme. To test this hypothesis, we first investigated the
BDDE induced changes in intrinsic tryptophan fluorescence of the α-glucosidase enzymes. As shown
in Figure 2, when excitated at 295 nm, BDDE had no obvious fluorescence emission, and contributed
negligible florescence interference. With the treatment of BDDE (0–100 μM), fluorescence intensity of
α-glucosidase was quenched gradually in a concentration dependent manner. These results established
that there is an interaction between BDDE and α-glucosidase, which induces a microenvironment
variation for Trp residues in α-glucosidase. Possibly, this variation hinders the active centre formation
and/or the binding of the substrates. The intrinsic fluorescence changes in α-glucosidase are similar to
those in lysozyme induced by bromophenol blue [19], and many other enzymatic inhibitors could also
induce fluorescence quenching of their target enzymes [20,21].
Figure 2. Intrinsic fluorescence spectra changes of α-glucosidase by BDDE. The enzyme
(2 μM) was incubated with specified concentrations of BDDE (0–100 μM) for 30 min at
37 °C. The excitation wavelength was 295 nm and emission spectra were acquired by
scanning from 300 to 400 nm.
2.3. BDDE Binding Reduced the Hydrophobicity of α-Glucosidase
The hydrophobic characterization and environmental sensitivity of bis-8-anilinonaphthalene-1-sulfonate
(bis-ANS) allow it to be widely used in measurement of protein surface hydrophobicity [22]. To study
the BDDE induced changes on the hydrophobicity of α-glucosidase, bis-ANS was employed and the
relative fluorescence intensities were shown in Figure 3. With increased concentrations of BDDE, the
enzyme-bis-ANS fluorescence was reduced in a concentration-dependent manner. These results
suggested that BDDE could reduce the hydrophobic surface of α-glucosidase. In our previous study,

Mar. Drugs 2011, 9
1558
reduced enzyme hydrophobicity were also observed when α-glucosidase bound to another inhibitor,
butyl-isobutyl-phthalate (BIP) [23]. Such inhibitor induced decreasing in the hydrophobicity supports
the notion that poor hydrophobic surface usually hinder the formation of active center in the
enzyme molecules.
Figure 3. Changes of α-glucosidase-bis-ANS complex fluorescence by BDDE.
α-Glucosidase (2 μM) was incubated for 30 min at 37 °C in the absence or presence of
specified concentrations of BDDE (0–100 μM). Bis-ANS (15 μM) was then added, and
fluorescence was measured after 15 min of incubation at 37 °C (excitation at 400 nm,
emission at 395–545 nm) using a Cary Eclipse fluorescence spectrophotometer.
2.4. BDDE Binding Influenced the Secondary Structures of α-Glucosidase
To study the effect of BDDE on the secondary structure of α-glucosidase, we measured the CD
spectra (190–250 nm) of α-glucosidase. As shown in Figure 4, BDDE (0–100 μM) triggered a minor
change in the CD spectrum of α-glucosidase, by increasing the α-helix (from 27.5% to 31.2%) and
decreasing the β-sheet content (from 39.4% to 31.6%, Table 2). This finding further supports the
proposition that BDDE binds to the enzyme and slightly changes the secondary conformation of
α-glucosidase. The increase in α-helix induced by BDDE is similar to α-glucosidase inhibitor
penicillamine, which also induces an increase in the α-helix content [24]. However, BDDE-induced
secondary structure changes are in contrast to other α-glucosidase inhibitors, for example,
curcuminoids analogs [7], BIP [23], and hydroxycoumarin derivatives [25], all of which decrease the
α-helix content in the enzyme molecule. The reason that these inhibitors induce contrary changes in the
α-helix content remains unknown. Nonetheless, it is clear that small conformational changes that
hamper active center formation or prevent substrates binding will inactivate the enzyme.

Mar. Drugs 2011, 9
Figure 4. CD spectra of α-glucosidase in the absence or presence of BDDE. α-glucosidase
1559
(2 μM, dissolved in 20 mM potassium phosphate buffer, pH 6.8) alone or incubated with
BDDE (0–100 μM) at 37 °C for 30 min. Spectra were acquired from 190 to 250 nm.
Table 2. The secondary structure content of α-glucosidase influenced by BDDE.
BDDE (μM)
α-Helix (%)
27.5
β-Sheet (%)
39.4
β-Turn (%)
Random (%)
0
25
50
100
7.1
5.6
8.3
7.1
26
30.3
28.9
31.2
33.8
34.7
31.6
30.3
28.2
30.2
The secondary structure content of α-glucosidase is predicted by program JWSSE (JASCO).
2.5. Protein 3D Structure Generation and Molecular Docking
Since the 3D structure of α-glucosidase is still unavailable, a homology model was developed based
on the crystal structure of S. cerevisiae oligo-1,6-glucosidase (Figure 5a). To explore the potential
protein ligand interactions, induced fit docking protocol was employed to dock BDDE into the glucose
binding site of the non-cognate homology structure. The BDDE molecule was successfully docked into
the active site with a highly favorable binding energy of −11.54 kcal/mol. The docked BDDE molecule
was completely buried in the α-glucosidase binding pocket with part of the molecule reaching the
catalytic center comprised of Asp214, Glu276 and Asp349 and overlapping with the position of
glucose in its complex crystal structure, which adequately explains the competitive nature of BDDE;
the remaining part of the molecule extended towards the protein surface. Using the same modeling and
docking protocols, we have previously studied BIP, a non-competitive α-glucosidase inhibitor, and
identified a novel ligand binding site located ~3 Å away from the catalytic center and close to the
protein surface [23]. Direct comparison of the docking results of BDDE and BIP through structural
alignment showed that there was no overlapping between the BIP and BDDE binding site, which

Mar. Drugs 2011, 9
1560
indicates potential non-competitive binding between these two ligands. Detailed structural analysis
(Figure 5b,c) suggested that the binding pocket of BDDE consisted of three areas: (1) a charged area
which is located near the active center and consists of charged residues including the negatively
charged catalytic triads and positively charged Arg439 and Arg212; (2) a polar area near protein
surface that consists of residue His279, His235 and Asn241; and (3) a hydrophobic area sandwiched
between residue Phe300, Ala278 and residue Phe158, Phe177, Phe157, Leu218. While the hydroxyl
groups at the two ends of BDDE molecule formed multiple hydrogen bonds separately with residues in
the charged and polar areas, the rest of the molecule were stabilized by hydrophobic interactions with
nearby residues. The observed multiple hydrogen bonds formed by the hydroxyl groups of BDDE are
consistent with the conclusion from previous studies that phenol moiety and hydroxyl groups in
hydroxycoumarin derivatives are critical for their inhibitory activities [25]. The identified
charged-hydrophobic-polar (C-H-P) binding pocket, which fits very well with the symmetrical feature
of BDDE, provides structural insight for design and development of novel α-glucosidase inhibitors.
However, further X-ray or NMR data are needed to verify this enzyme-inhibitor binding.
Figure 5. Structure model of BDDE-α-glucosidase complex. (a) Comparisons between
docked BDDE (green-carbon), BIP (blue-carbon) and the crystal structure of glucose
(yellow-carbon). Glucosidase is shown in grey ribbon, ligands are shown in solid sticks,
the triad residues (Asp214, Glu276 and Asp349) are shown in a CPK model;
(b) A close-up view of BDDE binding pocket. Molecular surface are shown and colored
according to their residue properties (red: negatively charged; blue: positively charged;
cyan: polar; green: hydrophobic); (c) The interactions between docked BDDE and nearby
residues. The triad residues are emphasized in a ball-and-stick model, and hydrogen bonds
are represented in dashed yellow lines.

Mar. Drugs 2011, 9
1561
3. Experimental Section
3.1. Materials
The baker’s yeast α-glucosidase (EC 3.2.1.20), p-nitrophenyl glycosides, and
bis-8-anilinonaphthalene-1-sulfonate (bis-ANS) were purchased from Sigma-Aldrich Chemical
(St. Louis, MO, USA). Other reagents were reagent grade available and purchased from
J&K Chemical Ltd. (Beijing, China).
3.2. Synthesis of BDDE
Procedures for the Preparation of Compounds 1 to 7
3-Bromo-4-hydroxy-5-methoxybenzaldehyde (2). To a solution of compound 1 (15.2 g) in
AcOH, Br2 (7 mL) was added at r.t. This mixture was stirred at r.t. for 2 h, and the compound 1
disappeared by TLC. Then the mixture was poured into cool water, and after filtration, the product
compound 2 was obtained as white solid (20.7 g, yield: 90%).
3-Bromo-4,5-dimethoxybenzaldehyde (3). To a solution of compound 2 (20.7 g) in CH3CN,
K2CO3 (19 g) and MeI (19 g) were added at r.t. Then this mixture was stirred at r.t. for 4 h. The
mixture was poured into water and white precipitation was showed and the compound 3 was obtained
after filtration as white solid (20 g, yield: 92 %).
2,3-Dibromo-4,5-dimethoxybenzaldehyde (4). Br2 (7 mL) was added to a solution of compound 3
(20 g) in AcOH at r.t. This mixture was stirred at 60 °C overnight. The TLC showed the compound 3
was consumed completely. Then the mixture was poured into cool water, and after filtration, the
product compound 4 was obtained as white solid (19.3 g, yield: 73%).
(2,3-Dibromo-4,5-dimethoxyphenyl)methanol (5). To a solution of compound 4 (19.0 g) in
MeOH, NaBH4 (4.52 g) was added at −20 °C. Then the mixture was warmed to r.t. slowly and stirred
for 3 h. After removing the solvent, the product compound 5 (13.3 g, yield: 70%) was obtained by
washing with water.
5,5′-Oxybis(methylene)bis(3,4-dibromo-1,2-dimethoxybenzene) (6). To a solution of compound 5
(13 g) in CH3CN, 4.54 g of TsCl and 2.41 g of Et3N were added at r.t. Then the mixture was refluxing
overnight. The TLC showed that compound 5 was completely consumed. The mixture was poured into
cool water, after filtration, the product compound 6 was obtained as white solid (6.56 g, yield: 52%).
Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (7). To a solution of compound 6 (6.0 g) in dry
°
CH2Cl2, BBr4/CH2Cl2 (2.0 M) solvent (20 mL) was added at 0 C. The mixture was warmed to r.t.
slowly and stirred for 4 h. Then, cool MeOH was added to quench the reaction. After removing the
solvent, the residue was separated by biotage® Flash chromatography (35% MeOH/H2O) to obtain
compound 7 (1.2 g, yield: 21.8%).

Mar. Drugs 2011, 9
1562
3.3. Intrinsic Fluorescence Measurements
α-Glucosidase (2 μM) was pretreated with certain concentrations of BDDE (0–100 μM) for 30 min
at 37 °C. The intrinsic fluorescence spectra (300–400 nm) were measured using a Cary Eclipse
fluorescence spectrophotometer (Varian Inc. USA) with the excitation wavelength was 295 nm.
3.4. Hydrophobic Analysis of α-Glucosidase Using Bis-ANS
α-Glucosidase (2 μM) was incubated at 37 °C for 30 min in the absence or presence of certain
concentrations of BDDE (0–100 μM). Bis-ANS (15 μM) was then added, and fluorescence was
measured after incubation at 37 °C for 15 min (λex = 400 nm, λem = 395–545 nm) using FlexStation II
microplate spectrofluorometer (Molecular Devices, USA).
3.5. Circular Dichroism Spectroscopy
CD spectra (190–250 nm) of α-glucosidase (2 μM) treated with different concentrations of BDDE
(0–100 μM) were measured with a JASCO 715 spectropolarimeter (JASCO, Tokyo, Japan). The
spectra were collected and corrected by subtraction of a blank containing 20 mM potassium phosphate
buffer (pH 6.8), reduction of noise, and smoothing. The program JWSSE (JASCO) was used
for estimation of the secondary structure percentage of α-glucosidase based on the method
of Yang et al. [26].
3.6. Molecular Modeling and Docking
Molecular modeling and docking studies were performed following the similar procedure as in our
previous study [23]. Briefly, a 3D homology model of Saccharomyces cerevisiae α-glucosidase was
built based on the complex crystal structure of α-D-glucose bound oligo-1,6-glucosidase (PDB ID:
3A4A) which shares 72% identical and 85% similar sequence with α-glucosidase. Sequence alignment
and structural model building were performed using the protein structure prediction suite Prime
(Schrödinger, New York, NY, USA). The constructed homology model was further optimized using
the protein preparation wizard of maestro 9.0 with OPLS2001 force field [27]. Docking studies were
then performed on the optimized model using the Induced-Fit-Docking (IFD) workflow [28], which is
capable of sampling dramatic side-chain conformational changes as well as minor changes in the
backbone structure. Specifically, the center of glucose molecule in the crystal structure was selected as
the centriod for ligand-docking, residues within 6 Å distance of glucose were set to be flexible. IFD
protocol was applied with default parameters to sample energetically reasonable side chains and to
eliminate conformations with steric crashes. The docked protein-ligand complexes were ranked
according to their IFD score made up of the protein-ligand interaction energy (GlideScore) and the
protein molecular mechanics energy (Prime energy) in an implicit solvent model [28].

Mar. Drugs 2011, 9
1563
4. Conclusions
In summary, BDDE has been synthesized and its interaction with α-glucosidase was studied. Our
results suggest that BDDE bind the enzyme molecule via both hydrogen-bonds and hydrophobic
interactions. BDDE binding hindered the substrate from accessing the catalytic site and induced
conformational changes on α-glucosidase, characterized by intrinsic fluorescence quenching,
hydrophobicity reduction, and α-helix content increase. This study provided structural insights
regarding the interaction between α-glucosidase and BDDE, and implied that BDDE could be
considered as novel template for the design of α-glucosidase inhibitors.
Acknowledgments
This work was supported by the innovative projects of the Chinese Academy of Sciences
(No. #KSCX2-YW-104 and KZCX2-YW-209).
References
1. Lefebvre, P.J.; Scheen, A.J. The use of acarbose in the prevention and treatment of
hypoglycaemia. Eur. J. Clin. Invest. 1994, 24 (Suppl. 3), 40–44.
2. Scott, L.J.; Spencer, C.M. Miglitol: A review of its therapeutic potential in type 2 diabetes
mellitus. Drugs 2000, 59, 521–549.
3. Campbell, L.K.; Baker, D.E.; Campbell, R.K. Miglitol: Assessment of its role in the treatment of
patients with diabetes mellitus. Ann. Pharmacother. 2000, 34, 1291–1301.
4. Krentz, A.J.; Bailey, C.J. Oral antidiabetic agents: current role in type 2 diabetes mellitus. Drugs
2005, 65, 385–411.
5. Nathan, D.M.; Buse, J.B.; Davidson, M.B.; Heine, R.J.; Holman, R.R.; Sherwin, R.; Zinman, B.
Management of hyperglycaemia in type 2 diabetes: A consensus algorithm for the initiation and
adjustment of therapy. A consensus statement from the American Diabetes Association and the
European Association for the Study of Diabetes. Diabetologia 2006, 49, 1711–1721.
6. Hsiao, S.H.; Liao, L.H.; Cheng, P.N.; Wu, T.J. Hepatotoxicity associated with acarbose therapy.
Ann. Pharmacother. 2006, 40, 151–154.
7. Du, Z.Y.; Liu, R.R.; Shao, W.Y.; Mao, X.P.; Ma, L.; Gu, L.Q.; Huang, Z.S.; Chan, A.S.
Alpha-glucosidase inhibition of natural curcuminoids and curcumin analogs. Eur. J. Med. Chem.
2006, 41, 213–218.
8. de Melo, E.B.; da Silveira Gomes, A.; Carvalho, I. alpha- and beta-glucosidase inhibitors:
Chemical structure and biological activity. Tetrahedron 2006, 62, 10277–10302.
9. Kwon, Y.I.; Apostolidis, E.; Shetty, K. In vitro studies of eggplant (Solanum melongena)
phenolics as inhibitors of key enzymes relevant for type 2 diabetes and hypertension.
Bioresour. Technol. 2008, 99, 2981–2988.
10. Tundis, R.; Loizzo, M.R.; Menichini, F. Natural products as alpha-amylase and alpha-glucosidase
inhibitors and their hypoglycaemic potential in the treatment of diabetes: An update. Mini Rev.
Med. Chem. 2010, 10, 315–331.

Mar. Drugs 2011, 9
1564
11. Ranilla, L.G.; Kwon, Y.-I.; Apostolidis, E.; Shetty, K. Phenolic compounds, antioxidant activity
and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension
of commonly used medicinal plants, herbs and spices in Latin America. Bioresour. Technol. 2010,
101, 4676–4689.
12. Kurihara, H.; Mitani, T.; Kawabata, J.; Takahashi, K. Two new bromophenols from the red alga
odonthalia corymbifera. J. Nat. Prod. 1999, 62, 882–884.
13. Kurihara, H.; Mitani, T.; Kawabata, J.; Takahashi, K. Inhibitory potencies of bromophenols from
Rhodomelaceae algae against α-glucosidase activity. Fish Sci. 1999, 65, 300–303.
14. Kim, K.Y.; Nam, K.A.; Kurihara, H.; Kim, S.M. Potent alpha-glucosidase inhibitors purified from
the red alga Grateloupia elliptica. Phytochemistry 2008, 69, 2820–2825.
15. Kim, K.Y.; Nguyen, T.H.; Kurihara, H.; Kim, S.M. Alpha-glucosidase inhibitory activity of
bromophenol purified from the red alga Polyopes lancifolia. J. Food. Sci. 2010, 75, H145–H150.
16. Xu, X.; Song, F.; Wang, S.; Li, S.; Xiao, F.; Zhao, J.; Yang, Y.; Shang, S.; Yang, L.; Shi, J.
Dibenzyl bromophenols with diverse dimerization patterns from the brown alga Leathesia nana.
J. Nat. Prod. 2004, 67, 1661–1666.
17. Xu, N.; Fan, X.; Yan, X.; Li, X.; Niu, R.; Tseng, C.K. Antibacterial bromophenols from the
marine red alga Rhodomela confervoides. Phytochemistry 2003, 62, 1221–1224.
18. Oh, K.B.; Jeon, H.B.; Han, Y.R.; Lee, Y.J.; Park, J.; Lee, S.H.; Yang, D.; Kwon, M.; Shin, J.;
Lee, H.S. Bromophenols as Candida albicans isocitrate lyase inhibitors. Bioorg. Med. Chem. Lett.
2010, 20, 6644–6648.
19. Yue, Q.; Niu, L.; Li, X.; Shao, X.; Xie, X.; Song, Z. Study on the interaction mechanism of
lysozyme and bromophenol blue by fluorescence spectroscopy. J. Fluoresc. 2008, 18, 11–15.
20. Qi, X.; Grabowski, G.A. Acid beta-glucosidase: intrinsic fluorescence and conformational
changes induced by phospholipids and saposin C. Biochemistry 1998, 37, 11544–11554.
21. Li, Y.; Gao, F.; Shan, F.; Bian, J.; Zhao, C. Study on the interaction between 3 flavonoid
compounds and alpha-amylase by fluorescence spectroscopy and enzymatic kinetics.
J. Food Sci. 2009, 74, C199–C203.
22. Hawe, A.; Sutter, M.; Jiskoot, W. Extrinsic fluorescent dyes as tools for protein characterization.
Pharm. Res. 2008, 25, 1487–1499.
23. Liu, M.; Zhang, W.; Qiu, L.; Lin, X. Synthesis of butyl-isobutyl-phthalate and its interaction with
alpha-glucosidase in vitro. J. Biochem. 2011, 149, 27–33.
24. Kim, J.H.; Kim, H.J.; Park, H.W.; Youn, S.H.; Choi, D.Y.; Shin, C.S. Development of
inhibitors against lipase and alpha-glucosidase from derivatives of monascus pigment.
FEMS Microbiol. Lett. 2007, 276, 93–98.
25. Shen, Q.; Shao, J.; Peng, Q.; Zhang, W.; Ma, L.; Chan, A.S.; Gu, L. Hydroxycoumarin
derivatives: Novel and potent alpha-glucosidase inhibitors. J. Med. Chem. 2010, 53, 8252–8259.
26. Yang, J.T.; Wu, C.S.; Martinez, H.M. Calculation of protein conformation from circular
dichroism. Methods Enzymol. 1986, 130, 208–269.
27. Price, M.L.P.; Ostrovsky, D.; Jorgensen, W.L. Gas-phase and liquid-state properties of esters,
nitriles, and nitro compounds with the OPLS-AA force field. J. Comput. Chem. 2001, 22,
1340–1352.

Mar. Drugs 2011, 9
1565
28. Sherman, W.; Day, T.; Jacobson, M.P.; Friesner, R.A.; Farid, R. Novel procedure for modeling
ligand/receptor induced fit effects. J. Med. Chem. 2006, 49, 534–553.
Samples Availability: Available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).